Fig. 2: 40S PICs are enriched on start codons due to loss of RPL11B and predict downstream translation. | Nature Communications

Fig. 2: 40S PICs are enriched on start codons due to loss of RPL11B and predict downstream translation.

From: 40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

Fig. 2

a Average ribosome footprint occupancy at start codons increased (~2×) for 40S data for cells lacking RPL11B. Footprints of 26–45 nt plotted by 5′ ends. b Comparison of 40S start codon peak levels (total footprints at the start codon) with LTM-treated 80S start codon peak levels (total footprints at the start codon) from Eisenberg et al. (2020)42 revealed a strong correlation (Pearson R2 = 0.61) showing that 40S peaks can be used to identify translation start sites. Each point represents the data for one gene. Footprint reads were quantitated after shifting 40S data by 14 nt and 80S data by 13 nt. c Data from 40S and 80S profiling data corresponding to the GRS1 and GRX2 genes. The 40S footprint occupancy peaks at cognate AUG and near-cognate UUG codons reveal the N-terminal extensions that are apparent in the 80S data. Gene annotations (DNA sequences and amino acids encoded by cognate tRNAs) are drawn to correspond to ribosome P sites. d Analysis of mitochondria that were purified from BY4741 cells expressing FLAG-tagged versions of either WT GRX2, GRX2-M1K, or GRX2-L19A from the sc expression vector YCplac33. Diagrams of the FLAG-tagged GRX2 alleles showing the three potential translation start sites and their mutations are shown on the left. TCA protein extracts from the purified mitochondria were subjected to western analysis using mouse monoclonal antibodies against FLAG (top blot) or VDAC1/Porin (mitochondrial control, bottom blot). Two amounts of extracts (1× and 2×) were loaded in each lane pair. The M1K mutation results in loss of expression of the mitochondrial outer membrane Grx2 isoform and the L19A mutation results in loss of expression of the mitochondrial matrix Grx2 isoform. Westerns were repeated twice on two independently grown cultures for each condition to ensure replicability. nt nucleotide, rpm reads per million, kDa kilodalton. Source data are provided as a Source Data file. See also Supplementary Fig. 2.

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